Method of treating ocular allergy

ABSTRACT

The topical use of 5,6,7-trihydroxyheptanoic acid and analogs alone or in combination with histamine antagonists and/or mast cell stabilizers is disclosed for the treatment of ocular allergy.

This application is a continuation of Ser. No. 11/593,714 filed Nov. 7,2006 now U.S. Pat. No. 7,687,539, which claims priority to U.S.Provisional Application, U.S. Ser. No. 60/734,194 filed Nov. 7, 2005.

The present invention is directed to the topical treatment of ocularallergic disorders, such as allergic conjunctivitis, giant papillaryconjunctivitis, vernal conjunctivitis, and atopic keratoconjunctivitis.In particular, the present invention is directed toward the topical useof 5,6,7-trihydroxyheptanoic acid and its analogs to treat ocularallergy.

BACKGROUND OF THE INVENTION

The eye, particularly the conjunctiva, has a relatively large number ofmast cells. When allergens are present they can bind to immunoglobulinson the surface of these mast cells and trigger their degranulation(breakdown). Degranulation releases mast cell components, includinghistamine, into the environment outside the mast cell. Through a varietyof mechanisms these components produce ocular surface inflammationresulting in itching, tearing, lid and conjunctival edema/redness, andphotophobia. This is frequently designated as an acute phase response,as is seen with seasonal allergic conjunctivitis and perennial allergicconjunctivitis. Topical ocular application of histamine receptorantagonists such as olopatidine or mast cell stabilizers such aslodoxamide are frequently used to alleviate these symptoms [for areview, see: Bielory et al., Drugs 2005, 65(2), 215-228].

As is the case in other allergic diseases, the acute phase response canprogress to a late phase response characterized by an influx ofeosinophils and neutrophils into the conjunctiva. In the associatedchronic allergic disease, exemplified by vernal keratoconjunctivitis,atopic keratoconjunctivitis, and giant papillary conjunctivitis, eyelidswelling and remodeling of the ocular surface tissues can occur. Insevere cases the patient experiences extreme discomfort and sustainsdamage to the ocular surface. For such instances there is no highlyeffective and safe treatment regimen. Although topical administration ofcorticosteroids is effective in severe cases, chronic use iscontraindicated due to an increased risk for the development ofcataracts and glaucoma [for a review, see: Ono and Abelson, J. AllergyClin. Immunol. 2005, 115(1), 118-122].

Lipoxin A₄ is an anti-inflammatory eicosanoid biosynthesized fromarachidonic acid, and is produced locally at inflammation sites via theinteraction of neutrophils with platelets or of other leukocytes withepithelial cells. Lipoxin A₄ is believed to act endogenously to resolveinflammation by inducing apoptosis and phagocytosis/clearance ofactivated leukocytes. Lipoxin A₄ binds to at least two receptors with nMaffinity. The first is the lipoxin A₄ cognate receptor, called ALXR.This is the same as the formyl peptide receptor FPRL-1. The secondreceptor is that for the cysteinyl leukotriene LTD₄. Lipoxins arethought to function as ALXR agonists and LTD₄ receptor antagonists[Fronert et al., Am. J. Pathol. 2001, 158(1), 3-8].

Several researchers have reported that administration of lipoxin A₄structural analogs inhibit allergen-induced eosinophil infiltration,decrease production of pro-inflammatory allergic mediators likecysteinyl leukotrienes, IL-5, and eotaxin, and reduce tissue edema inseveral animal models, including: a mouse model of allergic asthma [Levyet al., Nat. Med. 2002, 8(9), 1018-1023]; allergen-induced skininflammation in mice and guinea pigs [Schottelieus et al., J. Immun.2002, 169, 7063-7070]; and allergen-induced pleurisy in rats[Bandeira-Melo et al., J. Immun. 2000, 164(5), 2267-2271].

Lee et. al. have disclosed that compounds 1 and 2 inhibit LTB₄-inducedchemotaxis of neutrophils as potently as lipoxin A₄ [Lee et. al.,Biochemical and Biophysical Research Communications 1991, 180(3),1416-21]. As the authors' stated purpose was to investigate therelationship between this bioassay readout and the structure of lipoxinA₄ analogs that they synthesized, one conclusion could be that compounds1, 2, and lipoxin A₄ inhibit LTB₄-induced neutrophil chemotaxis by thesame mechanism, namely activation of the ALXR.

However, this theory may well be invalid. An essential experiment totest this theory would be to ascertain whether the chemotaxis inhibitioneffect for these three compounds could be blocked by a selective ALXRantibody or small molecule antagonist. This was not performed, since atthe time of Lee et al.'s disclosure neither the ALXR protein nor itsassociated mRNA had been sequenced [this was accomplished in 1994: J.Exp. Med. 1994, 180(1), 253-260]. An explanation for the neutrophilchemotaxis inhibition displayed by 1, 2, and lipoxin A₄ which is equallyconsistent with this disclosure would be that 1 and 2 act vialeukotriene B₄ receptor antagonism while lipoxin A₄ acts via ALXRagonism and/or perhaps antagonism at the leukotriene D₄ (LTD₄) receptor[Gronert et al., Am. J. Path. 2000, 158(1), 3-9]. Furthermore it isknown that the biological activity of lipoxin A₄ is critically dependenton the presence of a hydroxyl at position 15; oxidation to the carbonyl[Petasis et al., Prostaglandins Leukot. Essent. Fatty Acids 2005,73(3-4), 301-321] or replacement with a hydrogen [Jozsef et al., Proc.Natl. Acad. Sci. USA 2002, 99(20), 13266-13271] greatly diminishesbiological activity. However 1 and 2 lack this hydroxyl, indeed theylack any atoms at all beyond the primary hydroxyl group of their triolarray. To the best of our knowledge there have been no subsequentreports on the biological activities of either 1 or 2. Thus absentreceptor-linked functional data, one skilled in the art could reasonablydoubt that these compounds' inhibition of LTB₄-induced neutrophilchemotaxis is due to ALXR agonism.

SUMMARY OF THE INVENTION

The present invention is directed to methods for the topical treatmentof ocular allergy, including seasonal and perennial allergicconjunctivitis, vernal keratoconjunctivitis, atopickeratoconjunctivitis, and giant papillary conjunctivitis. According tothe methods of the present invention, a 5,6,7-trihydroxyheptanoic acidor analog is topically administered to a patient, alone or incombination with a histamine receptor antagonist and/or a mast celldegranulation inhibitor, such as olopatidine and emedastine. The5,6,7-trihydroxyheptanoic acid or analog is administered in anophthalmic composition dosed topically to a patient's eye.

DETAILED DESCRIPTION OF THE INVENTION

Unless indicated otherwise, all component amounts are presented on a %(w/v) basis.

According to the methods of the present invention, a compositioncomprising a compound of formula I alone or in combination with ahistamine receptor antagonist and/or a mast cell degranulation inhibitoris topically administered to a mammal in need thereof:

wherein

-   R¹ is C₂H₅, CO₂R, CONR²R³, CH₂OR⁴, or CH₂NR⁵R⁶, where:    -   R is H, C₁₋₆ straight chain or branched alkyl, C₃₋₆ cycloalkyl,        or phenyl,    -   or R¹ is a carboxylate salt of formula CO₂ ⁻R⁺, where R⁺ is Li⁺,        Na⁺, K⁺,    -   or an ammonium moiety of formula ⁺NR¹⁰R¹¹R¹²R¹³;    -   R², R³ are independently H, C₁₋₆ alkyl, C₃₋₆ cycloalkyl, benzyl,        phenyl, OH, OCH₃, or OC₂H₅, provided that at most only one of        R², R³ is OH, OCH₃, or OC₂H₅;    -   R⁴ is H, C(O)R¹⁴, C₁₋₆ alkyl, C₃₋₆ cycloalkyl, benzyl, or        phenyl;    -   R⁵, R⁶ are independently H, C(O)R¹⁴, C₁₋₆ alkyl, C₃₋₆        cycloalkyl, benzyl, phenyl, OH, OCH₃, or OC₂H₅, provided that at        most only one of R², R³ is OH, OCH₃, or OC₂H₅;    -   R⁷, R⁸, and R⁹ are independently H, CH₃, C₂H₅, C(O)R¹⁴, or        CO₂R¹⁵;    -   or R⁷ and R⁸ or R⁸ and R⁹ together constitute a carbonyl group        (C═O), thus forming a cyclic carbonate;    -   or OR⁸R¹ together form a cyclic ester (a lactone);    -   R¹⁰-R¹³ are independently H or C₁₋₆ alkyl, each alkyl group        optionally bearing an OH or OCH₃ substituent;    -   R¹⁴ is H, C₁₋₆ alkyl, C₃₋₆ cycloalkyl, benzyl, or phenyl;    -   R¹⁵ is C₁₋₆ alkyl, C₃₋₆ cycloalkyl, benzyl, or phenyl; and        indicates that the OR⁹ substituent can be arranged to afford the        R or S absolute configuration:

Preferred compounds of formula I are those wherein:

-   -   R¹ is C₂H₅, CO₂R, CH₂OR⁴, or a carboxylate salt of formula CO₂        ⁻R⁺;    -   R⁺ is Li⁺, Na⁺, K⁺, or NH₄ ⁺;    -   R is H, CH₃, C₂H₅, n-C₃H₇, or i-C₃H₇;    -   R⁴ is H, COCH₃, or CH₃; and    -   R⁷, R⁸, R⁹ are independently H, CH₃, or CH₃CO;    -   or R⁷ and R⁸ or R⁸ and R⁹ together constitute a carbonyl group        (C═O), thus forming a cyclic carbonate;    -   or OR⁸R¹ together form a cyclic ester (a lactone).

Among the especially preferred are compounds 1-6. Compound 1 iscommercially available from Biomol Research Laboratories, PlymouthMeeting, Pa., and compound 2 can be prepared as detailed in Lee et. al.,Biochemical and Biophysical Research Communications 1991, 180(3),1416-21. Compounds 3-6 can be prepared as described in examples 1-4below.

EXAMPLE 1 Synthesis of Compound 3

A solution of methyl ester 1 (20 mg, 0.104 mmol) in MeOH (2.1 mL)containing 1 M LiOH (0.5 mL, 0.5 mmol) was heated in a microwave heaterat 120° C. for 6 minutes. The reaction was concentrated and the residuewas chromatographed on a 10 mm diameter×18 cm tall C18 reverse-phasesilica gel column eluting with 7:3 v:v 0.05 M HCl:acetonitrile to afforda crude white solid after concentration (40.9 mg). The solid was rinsedwith hot CH₃CN (2×2 mL) and the filtrate was concentrated to affordlactone 3 (7.8 mg, 47%). ¹³C NMR (150 MHz, dmso-d₆) δ 171.12 (C), 79.86(CH), 72.44 (CH), 62.03 (CH₂), 29.39 (CH₂), 21.67 (CH₂), 17.55 (CH₂).

EXAMPLE 2 Synthesis of Compound 4

A solution of methyl ester 1 in aqueous MeOH is heated to reflux in thepresence of 3 equivalents of lithium hydroxide. After 6 h the reactionis cooled to room temperature and the pH of the solution is adjusted to6 by the addition of 70-9 mesh sulfonic acid resin MP (commerciallyavailable from Novabiochem/EMD Biosciences, 10394 Pacific Center Court,San Diego, Calif. 92121). The solution is filtered through a 0.2 μMpoly-terfluoroethylene syringe filter and concentrated to afford thelithium carboxylate 4 as a white solid. ¹H NMR (D₂O, 400 MHz) δ3.69-3.64 (m, 1H), 3.55-3.47 (m, 3H), 2.16-2.12 (m, 2H), 1.67-1.64 (m,1H), 1.54-1.48 (m, 2H), 1.38-1.34 (m, 1H). ¹³C NMR (D₂O, 100 MHz) δ183.46 (C), 74.61 (CH), 71.67 (CH), 62.49 (CH₂), 37.26 (CH₂), 31.55(CH₂), 22.04 (CH₂).

EXAMPLE 3 Synthesis of Compound 8

2-deoxy-D-ribose is converted to the acetonide-protected lactol 10 bytreatment with 2-methoxypropene and catalytic pyridiniump-toluenesulfonate (PPTS) in ethyl acetate. Wittig reaction withPh₃P═CHCO₂Et in THF in the presence of catalytic benzoic acid affordsenoate 11, which is reduced to 12 under a hydrogen atmosphere in thepresence of catalytic Pd/C in ethanol. Deprotection of 12 using 0.1 NHCl in ethanol for 5 minutes, followed by quenching with aqueous NaHCO₃,affords 8 after silica gel chromatographic purification.

EXAMPLE 4 Synthesis of Compound 9

Wittig reaction of lactol 10 with Ph₃P═CHCO₂Et in THF in the presence ofcatalytic benzoic acid affords enoate 13, which is reduced to 14 under ahydrogen atmosphere in the presence of catalytic Pd/C in isopropanol.Deprotection of 14 using 0.1 N HCl in isopropanol for 5 minutes,followed by quenching with aqueous NaHCO₃, affords 9 after silica gelchromatographic purification.

According to the methods of the present invention, a compound of formulaI is administered in a pharmaceutically acceptable carrier for topicalophthalmic administration. The compositions are formulated in accordancewith methods known in the art. The compositions may contain more thanone compound of formula I. Additionally, the compositions may contain asecond drug, other than a compound of formula I.

Compound I was evaluated in a mouse model of late-phase allergy asoutlined in example 5 below.

EXAMPLE 5 Activity of Compound 1 in a Mouse Late-Phase Allergy Model

Methods

Active Sensitization and Induction of Allergic Conjunctivitis (AC)

Female BALB/c mice, 6 to 9 months old (Charles River Labs), were given asingle intraperitoneal injection of 100 μg chicken ovalbumin (OVA;Sigma) which had been absorbed to 5 mg of alum (Pierce Chemical) as anadjuvant or 5 mg alum only (unsensitized group). On day 14 aftersensitization, all mice were challenged with a single topical drop O.D.of 1 mg OVA dissolved in 5 μl PBS.

Drugs or vehicles were administered to mice as a single 5 μl drop O.D.at 60 min before challenge and again at 16 hrs after challenge (BIDdosing). Mice were euthanized at 24 hrs after challenge.

EPO Activity Assay

Mice were euthanized at 24 hrs after topical challenge and upper andlower eyelids containing palpebral conjunctiva were excised andimmediately frozen on dry ice. Samples were weighed frozen and thenthawed and homogenized on ice in 2 ml of 50 mM HEPES buffer, pH 6.5.Samples were pelleted at 4000 rpm for 20 min at 4° C. and supernatantswere discarded. To each pellet, 1 ml of 0.5% cetyltrimethylammoniumchloride (CTAC) was added and samples were vortexed vigorously. Sampleswere then subjected to three freeze-thaw cycles between −80° C. and 37°C. After final thaw, samples were sonicated on ice for 15-30 sec andpelleted at 4000 rpm for 20 min at 4° C. EPO activity assay wasperformed on supernatants.

For the EPO activity assay, 75 μl of each sample were added intriplicate to wells of a 96-well clear flat-bottomed microplate. 75 μlof EPO substrate solution [6 mM o-phenylenediamine (OPD), 8.8 mM H₂O₂,and 6 mM KBr in 50 mM HEPES, pH 6.5] were then added to each well usinga multichannel pipetter. The reaction was allowed to run for 3 min andwas stopped by addition of 150 μl of 4M H₂SO₄. Optical density at 490 nm(OD₄₉₀) was read on a Bio-Tek Synergy. HT plate reader. EPO standardswere prepared by ½ serial dilutions from 1000 to 15.6 ng/ml of human EPOprotein (Calbiochem) in 50 mM HEPES, pH 6.5, with 6 mM KBr. 75 μltriplicates of each standard were used on each run of the assay. Linearregression was determined for the resulting plot of [EPO] vs OD490.Final [EPO] in each sample was determined by solving for x in theequation y=m*x+b, where y=OD490, m=x-intercept, and b=slope.

Statistical Methods

Groups of means were compared using Student's unpaired t-test orDunnett's t-test where appropriate. Means were considered to besignificantly different at P<0.05. Data are expressed as mean±standarddeviation.

Results

All three doses of 1 tested (0.001%, 0.01%, and 0.1% solutions)significantly inhibited conjunctival EPO activity at 24 hrspost-challenge with comparable efficacy to that of 0.1% dexamethasone(Table 1). Efficacy for each concentration of 1 was comparable 0.1%dexamethasone.

TABLE 1 Conjunctival EPO activity at 24 hrs post-challenge EPO Activity(ng/ml/mg Test Item Dose tissue) ± S.D.^(a) —^(b) —  3.0 ± 0.5 Vehicle —10.5 ± 3.5 dexamethasone 0.1%  4.0 ± 0.5^(c) compound 1 0.001%   5.2 ±2.5^(c,d) compound 1 0.01%   4.8 ± 1.2^(c,d) compound 1 0.1%  3.2 ±1.0^(c,d) ^(a)S.D. = standard deviation. ^(b)unsensitized mouse. ^(c)p <0.05 compared to vehicle-treated group by Dunnet's t-test. ^(d)Notstatistically different from dexamethasone-treated group (p > 0.05compared to dexamethasone-treated group by Dunnet's t-test).

The compositions of the present invention contain a pharmaceuticallyeffective amount of a compound of formula I. As used herein, “apharmaceutically effective amount” means an amount sufficient to reduceor eliminate allergic conjunctivitis symptoms. Generally, thecompositions of the present invention will contain from 0.000001 to 1%of a compound of formula I. Preferably, the compositions of the presentinvention will contain from 0.00003 to 0.01% of a compound of formula I.

The compositions administered according to the present invention mayalso include various other ingredients, including but not limited tosurfactants, tonicity agents, buffers, preservatives, co-solvents andviscosity building agents.

Various tonicity agents may be employed to adjust the tonicity of thecomposition, preferably to that of natural tears for ophthalmiccompositions. For example, sodium chloride, potassium chloride,magnesium chloride, calcium chloride, dextrose and/or mannitol may beadded to the composition to approximate physiological tonicity. Such anamount of tonicity agent will vary, depending on the particular agent tobe added. In general, however, the compositions will have a tonicityagent in an amount sufficient to cause the final composition to have anophthalmically acceptable osmolality (generally about 150-450 mOsm,preferably 250-350 mOsm).

An appropriate buffer system (e.g., sodium phosphate, sodium acetate,sodium citrate, sodium borate or boric acid) may be added to thecompositions to prevent pH drift under storage conditions. Theparticular concentration will vary, depending on the agent employed.Preferably, however, the buffer will be chosen to maintain a target pHwithin the range of pH 5.5-8.

Topical ophthalmic products are typically packaged in multidose form.Preservatives are typically required to prevent microbial contaminationduring use. Suitable preservatives include: benzalkonium chloride,chlorobutanol, benzododecinium bromide, methyl paraben, propyl paraben,phenylethyl alcohol, edetate disodium, sorbic acid, polyquaternium-1, orother agents known to those skilled in the art. Such preservatives aretypically employed at a level of from 0.001 to 1.0% w/v. Unit dosecompositions of the present invention will be sterile, but typicallywill not contain a preservative and will be unpreserved.

Generally, 1-2 drops of such compositions will be administered from onceto many times per day.

Representative eye drop formulations are provided below in Examples 6and 7 for treating allergic conjunctivitis.

EXAMPLE 6

Ingredient Concentration (% w/v) Compound of formula I 0.00001 to 1Ethanol 0.03-0.2  Boric Acid 0.1-0.3 Polyoxyl 40 Stearate 0.1 EdetateDisodium 0.01 Polyquaternium 1 0.001 NaOH/HCl q.s. to pH 6-8 PurifiedWater q.s. to 100%

EXAMPLE 7

Ingredient Concentration (% w/v) Compound of formula I 0.00003 to 0.01Hydroxypropyl methylcellulose 0.1-0.5 Dextran 70 0.1 Sodium Chloride 0.8Potassium Chloride 0.12 Dibasic Sodium Phosphate 0.025 Edetate Disodium0.01 Polyquaternium-1 0.001-0.005 NaOH/HCl q.s. to pH 6-8 Purified Waterq.s. to 100

This invention has been described by reference to certain preferredembodiments; however, it should be understood that it may be embodied inother specific forms or variations thereof without departing from itsspecial or essential characteristics. The embodiments described aboveare therefore considered to be illustrative in all respects and notrestrictive, the scope of the invention being indicated by the appendedclaims rather than by the foregoing description.

1. A method for the treatment of ocular allergy in a mammal, whichcomprises topically administering to the eye of the mammal a compositioncomprising a pharmaceutically acceptable carrier and a pharmaceuticallyeffective amount of a compound of formula I:

wherein R¹ is CONR²R³ or CH₂NR⁵R⁶, where: R², R³ are independently H,C₁₋₆ alkyl, C₃₋₆ cycloalkyl, benzyl, phenyl, OH, OCH₃, or OC₂H₅,provided that at most only one of R², R³ is OH, OCH₃, or OC₂H₅; R⁵, R⁶are independently H, C(O)R¹⁴, C₁₋₆ alkyl, C₃₋₆ cycloalkyl, benzyl,phenyl, OH, OCH₃, or OC₂H₅, provided that at most only one of R², R³ isOH, OCH₃, or OC₂H₅; R⁷, R⁸, and R⁹ are independently H, CH₃, C₂H₅,C(O)R¹⁴, or CO₂R¹⁵; or R⁷ and R⁸ or R⁸ and R⁹ together constitute acarbonyl group (C═O), thus forming a cyclic carbonate; or OR⁸R¹ togetherform a cyclic ester (a lactone); R¹⁴ is H, C₁₋₆ alkyl, C₃₋₆ cycloalkyl,benzyl, or phenyl; R¹⁵ is C₁₋₆ alkyl, C₃₋₆ cycloalkyl, benzyl, orphenyl; and

indicates that the OR⁹ substituent can be arranged to afford the R or Sabsolute configuration:


2. The method of claim 1 wherein for the compound of formula I: R⁷, R⁸,R⁹ are independently H, CH₃, or CH₃CO; or R⁷ and R⁸ or R⁸ and R⁹together constitute a carbonyl group (C═O), thus forming a cycliccarbonate; or OR⁸R¹ together form a cyclic ester (a lactone).
 3. Themethod of claim 2 wherein the compound of formula I has theconfiguration:


4. The method of claim 2, wherein the compound of formula I has theconfiguration:


5. The method of claim 3, wherein a compound of formula I is used totreat seasonal or perennial allergic conjunctivitis.
 6. The method ofclaim 3, wherein a compound of formula I is used to treat vernalkeratoconjunctivitis, atopic keratoconjunctivitis, or giant papillaryconjunctivitis.
 7. The method of claim 6, wherein the compound offormula I is used in combination with a histamine receptor antagonistand/or a mast cell stabilizer.
 8. The method of claim 7, wherein thehistamine receptor antagonist and/or mast cell stabilizer is selectedfrom the group consisting of: emedastine; levocabastine; mequitazine;chlorpheniramine; brompheniramine; astemizole; cetirizine; terfenadine;rocastine; loratadine; desloratadine [that is,8-chloro-6,11-dihydro-11-(4-piperidinylidene)-5H-benzo[5,6]cyclohepta[1,2-b]pyridine];5-[2-[4-bis(4-fluorophenyl)hydroxymethyl-1-piperidinyl]ethyl]-3-methyl-2-oxazolidinoneethanedioate; pyrilamine; clemastine; azelastine; epinastine; ketotifen;olopatadine; mapinastine; lodoxamide; cromolyn sodium; and nedocromildisodium salt.
 9. The method of claim 1, wherein the pharmaceuticallyeffective amount of the compound of formula I is from 0.00003 to 0.01%(w/v).
 10. The method of claim 9, wherein the pharmaceuticallyacceptable carrier comprises one or more ingredients selected from thegroup consisting of surfactants; tonicity agents; buffers;preservatives; co-solvents; and viscosity building agents.